Fig 1.
Schematic representation of the experimental paradigm.
[Abbreviations: DA, dopamine; •OH, hydroxyl radical; GSH, reduced glutathione; SOD, superoxide dismutase; CAT, catalase; TH-IR, tyrosine hydroxylase-immunoreactivity].
Fig 2.
Effect of high cholesterol diet on (A) body weight, (B) serum total cholesterol, and cholesterol level in (C-D) liver and (F-G) brain (striatum).
Mice were fed with high cholesterol diet (HCD) or standard diet (control, CS) for 14 weeks. Body weight (in gram) was measured two weeks apart from the start of treatment till 14th weeks. On the last day of diet, blood was collected by cardio-punctured method and serum total cholesterol was estimated by enzymatic method. Accumulation of cholesterol in liver and striatum region of brain was estimated from fixed tissues by Schultz’s method. Optical density of characteristic greenish-blue colour for cholesterol in liver (E) and striatum (H) was measured using ImageJ software. The results given are mean ± S.E.M. *p ≤0.05 as compared to CS (n = 8).
Fig 3.
Effect of hypercholesterolemia on Parkinsonian motor behavior.
In the last week of the 14 week treatment period, all the groups of animals were tested for (A) akinesia (B) catalepsy and (C) Swim test. The results are given as mean ± S.E.M. *p ≤ 0.05 as compared to control (CS) and #p ≤ 0.05 as compared to MPTP alone treated group (n = 6).
Fig 4.
Effect of hypercholesterolemia on striatal dopamine level in Parkinsonian mice.
Mice were sacrificed by decapitation on the seventh day following the first dose of MPTP. Striatal dopamine content was analyzed by HPLC-ECD system. Hypercholesterolemia exaggerates striatal dopamine depletion in Parkinsonian mice. The results are given as mean ± S.E.M. *p ≤ 0.05 as compared to control (CS) and #p ≤ 0.05 as compared to MPTP alone treated group (n = 6).
Fig 5.
Effects of hypercholesterolemia on tyrosine hydroxylase (TH)-immunoreactivity in striatum (NCP) and TH-positive nigral (SN) neurons in MPTP-treated mice.
Representative NCP (A-D) and SN (E-H) photographs from CS, HCD, MPTP and HCD+MPTP (left to right) groups showing TH-immunoreactivity. Quantification of relative density of TH-immunostaining in NCP (I) and neuronal count in SN (J) analyzed using ImageJ software. Hypercholesterolemia aggravates nigral TH-positive neuronal loss in MPTP-treated mice. Results are expressed as mean ± SEM. *p ≤ 0.05 as compared to control (CS) and #p ≤ 0.05 as compared to MPTP alone treated group (n = 5).
Fig 6.
Effect of hypercholesterolemia on nigrostriatal mitochondrial complex-I activity in Parkinsonian mice.
Mitochondrial complex-I activity was analyzed in the (A) striatum and (B) substantia nigra regions of brain by employing a spectrophotometric procedure using NADH as substrate. Results given are mean ± SEM of nmol of NADH oxidized/min/mg protein. *p ≤ 0.05 as compared to control (CS) and #p ≤ 0.05 as compared to MPTP alone treated group (n = 4).
Fig 7.
Effect of hypercholesterolemia on nigrostriatal mitochondrial complex-II activity in Parkinsonian mice.
(A-D) representative striatal (NCP) sections and (E-H) substantia nigral (SN) sections were processed for mitochondrial complex-II activity by employing histoenzymology. The marked region in the photographs (E-H) represents substantia nigra pars compacta (SNpc) region [59]. Optical density of serial sections of (I) NCP and (J) SN was analyzed using ImageJ software. The results are given as mean ± S.E.M. *p ≤ 0.05 as compared to control (CS) and #p ≤ 0.05 as compared to MPTP alone treated group (n = 4).
Fig 8.
Effect of hypercholesterolemia on nigrostriatal mitochondrial complex-III activity in Parkinsonian mice.
(A-D) representative striatal (NCP) sections and (E-H) substantia nigral (SN) sections were processed for mitochondrial complex-III activity by employing histoenzymology. The marked region in the photographs (E-H) represents substantia nigra pars compacta (SNpc) region [59]. Optical density of serial sections of (I) NCP and (J) SN was analyzed using ImageJ software. The results are given as mean ± S.E.M. *p ≤ 0.05 as compared to control (CS) and #p ≤ 0.05 as compared to MPTP alone treated group (n = 4).
Fig 9.
Effect of hypercholesterolemia on hydroxyl radical (•OH) generation in (A,C,E) striatum and (B,D,F) substantia nigra regions of brain of Parkinsonian mice.
Animals were injected with salicylic acid (100 mg/kg) and sacrificed two hours post injection on the last day of treatment. 2,3- and 2,5-dihydroxy benzoic acid (DHBA; •OH adducts of salicylate) formed were measured from homogenates of NCP and SN by employing a sensitive HPLC-ECD method. Data are expressed as pmol/mg tissue and represented as mean ± S.E.M. *p ≤ 0.05 as compared with control and #p ≤ 0.05 as compared with MPTP alone treated group (n = 5).
Fig 10.
Effect of hypercholesterolemia on nigrostriatal reduced glutathione (GSH) level in Parkinsonian mice.
(A) Striatum and (B) substantia nigra regions of brain were used for estimating GSH levels by employing a sensitive HPLC-ECD system. Data are expressed as nmol/mg tissue and represented as mean ± S.E.M. *p ≤ 0.05 as compared with control and #p ≤ 0.05 as compared with MPTP alone treated group (n = 5).
Fig 11.
Effect of hypercholesterolemia on nigrostriatal antioxidant enzymes activity in Parkinsonian mice.
(A-B) superoxide dismutase (SOD) and (C-D) catalase (CAT) activity measured from the cytosolic fraction of (A) striatum and (B) substantia nigra regions of brain. SOD activity was analyzed by employing pyrogallol oxidation method. One unit of the SOD activity is defined as 50% inhibition/min/mg protein. CAT activity was analyzed by monitoring the disappearance of hydrogen peroxide in presence of the enzyme. Specific activity of CAT is described as change in absorbance at 240 nm/min/mg protein. The results are given as mean ± S.E.M. *p ≤ 0.05 as compared to control (CS) and #p ≤ 0.05 as compared to MPTP alone treated group (n = 5).